The ESX-1 (ESAT-6 system 1) secretion system is a key virulence determinant in both mycobacteria and Gram-positive pathogens. There is a fundamental gap in understanding the molecular mechanisms of this secretion system because all of the genes required for ESX-1 function have not been identified. The absence of this information is because a saturating genetic screen for requirements of ESX-1 secretion has never been performed due to technical limitations in the direct, high throughput measurement of ESX-1 function. However, the acquisition of such knowledge is critical to the basic understanding of mycobacterial virulence. The long-term goal is to understand the basic molecular mechanisms of protein secretion systems that underlie mycobacterial pathogenesis. The objective of this proposal is to attain the primary step toward this goal by identifying all of the genes required for ESX-1 secretion in pathogenic mycobacteria. The hypothesis is that this can be accomplished using a direct saturating genetic screen to monitor ESX-1 function. A proteomics based method that allows direct, high throughput monitoring of secretion of mycobacterial ESX-1 substrates was devised and validated in the applicant's laboratory. The rationale that underlies this proposal is that the knowledge amassed in the completion of this project will greatly advance the understanding of this important virulence pathway. Therefore, the following specific aim has been proposed: To conduct a direct, saturating genetic screen to comprehensively identify the components of the ESX-1 secretion system in pathogenic mycobacteria. A method using whole colony MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry to directly monitor ESX-1 protein secretion in bacteria has been established as feasible in the applicant's hands. This method will be used to conduct large scale screening of a Mycobacterium marinum transposon library to identify colonies that do not secrete ESX-1 substrates. The proposed approach is innovative because it represents a direct departure from the status quo and will overcome the current technical hurdle to identifying all components of the ESX-1 system. This contribution will be significant because it is expected to result in a great advance in understanding the basic biology of ESX-1 secretion.